The de novo biosynthesis of glycogen is catalyzed by glycogenin, a self-glucosylating protein primer. To date, the role of glycogenin in regulating glycogen metabolism and the attainment of maximal glycogen levels in skeletal muscle are unknown. We measured glycogenin activity after enzymatic removal of glucose by α-amylase, an indirect measure of glycogenin amount. Seven male subjects performed an exercise and dietary protocol that resulted in one high-carbohydrate leg (HL) and one low-carbohydrate leg (LL) before testing. Resting muscle biopsies were obtained and analyzed for total glycogen, proglycogen (PG), macroglycogen (MG), and glycogenin activity. Results showed differences ( P < 0.05) between HL and LL for total glycogen (438.0 ± 69.5 vs. 305.7 ± 57.4 mmol glucosyl units/kg dry wt) and PG (311.4 ± 38.1 vs. 227.3 ± 33.1 mmol glucosyl units/kg dry wt). A positive correlation between total muscle glycogen content and glycogenin activity ( r = 0.84, P < 0.001) was observed. Similar positive correlations ( P < 0.05) were also evident between both PG and MG concentration and glycogenin activity (PG, r = 0.82; MG, r = 0.84). It can be concluded that glycogenin does display activity in human skeletal muscle and is proportional to glycogen concentration. Thus it must be considered as a potential regulator of glycogen synthesis in human skeletal muscle.